Genome-wide identification of CaWD40 proteins reveals the involvement of a novel complex (CaAN1-CaDYT1-CaWD40-91) in anthocyanin biosynthesis and genic male sterility in Capsicum annuum.

BACKGROUND: The WD40 domain, one of the most abundant in eukaryotic genomes, is widely involved in plant growth and development, secondary metabolic biosynthesis, and mediating responses to biotic and abiotic stresses. WD40 repeat (WD40) protein has been systematically studied in several model plants but has not been reported in the Capsicum annuum (pepper) genome. RESULTS: Herein, 269, 237, and 257 CaWD40 genes were identified in the Zunla, CM334, and Zhangshugang genomes, respectively. CaWD40 sequences from the Zunla genome were selected for subsequent analysis, including chromosomal localization, phylogenetic relationships, sequence characteristics, motif compositions, and expression profiling. CaWD40 proteins were unevenly distributed on 12 chromosomes, encompassing 19 tandem duplicate gene pairs. The 269 CaWD40s were divided into six main branches (A to F) with 17 different types of domain distribution. The CaWD40 gene family exhibited diverse expression patterns, and several genes were specifically expressed in flowers and seeds. Yeast two-hybrid (Y2H) and dual-luciferase assay indicated that CaWD40-91 could interact with CaAN1 and CaDYT1, suggesting its involvement in anthocyanin biosynthesis and male sterility in pepper. CONCLUSIONS: In summary, we systematically characterized the phylogeny, classification, structure, and expression of the CaWD40 gene family in pepper. Our findings provide a valuable foundation for further functional investigations on WD40 genes in pepper.

Transcriptomic Insights into Salt Stress Response in Two Pepper Species: The Role of MAPK and Plant Hormone Signaling Pathways.

Salt stress imposes significant plant limitations, altering their molecular, physiological, and biochemical functions. Pepper, a valuable herbaceous plant species of the Solanaceae family, is particularly susceptible to salt stress. This study aimed to elucidate the physiological and molecular mechanisms that contribute to the development of salt tolerance in two pepper species (Capsicum baccatum (moderate salt tolerant) and Capsicum chinense (salt sensitive)) through a transcriptome and weighted gene co-expression network analysis (WGCNA) approach to provide detailed insights. A continuous increase in malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels in C. chinense and higher activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) in C. baccatum indicated more tissue damage in C. chinense than in C. baccatum. In transcriptome analysis, we identified 39 DEGs related to salt stress. Meanwhile, KEGG pathway analysis revealed enrichment of MAPK and hormone signaling pathways, with six DEGs each. Through WGCNA, the ME.red module was identified as positively correlated. Moreover, 10 genes, A-ARR (CQW23_24856), CHIb (CQW23_04881), ERF1b (CQW23_08898), PP2C (CQW23_15893), ABI5 (CQW23_29948), P450 (CQW23_16085), Aldedh1 (CQW23_06433), GDA (CQW23_12764), Aldedh2 (CQW23_14182), and Aldedh3 (CQW23_11481), were validated by qRT-PCR. This study provides valuable insights into the genetic mechanisms underlying salt stress tolerance in pepper. It offers potential targets for future breeding efforts to enhance salt stress resilience in this crop.

Within-plant genetic drift to control virus adaptation to host resistance genes.

Manipulating evolutionary forces imposed by hosts on pathogens like genetic drift and selection could avoid the emergence of virulent pathogens. For instance, increasing genetic drift could decrease the risk of pathogen adaptation through the random fixation of deleterious mutations or the elimination of favorable ones in the pathogen population. However, no experimental proof of this approach is available for a plant-pathogen system. We studied the impact of pepper (Capsicum annuum) lines carrying the same major resistance gene but contrasted genetic backgrounds on the evolution of Potato virus Y (PVY). The pepper lines were chosen for the contrasted levels of genetic drift (inversely related to Ne, the effective population size) they exert on PVY populations, as well as for their contrasted resistance efficiency (inversely related to the initial replicative fitness, Wi, of PVY in these lines). Experimental evolution was performed by serially passaging 64 PVY populations every month on six contrasted pepper lines during seven months. These PVY populations exhibited highly divergent evolutionary trajectories, ranging from viral extinctions to replicative fitness gains. The sequencing of the PVY VPg cistron, where adaptive mutations are likely to occur, allowed linking these replicative fitness gains to parallel adaptive nonsynonymous mutations. Evolutionary trajectories were well explained by the genetic drift imposed by the host. More specifically, Ne, Wi and their synergistic interaction played a major role in the fate of PVY populations. When Ne was low (i.e. strong genetic drift), the final PVY replicative fitness remained close to the initial replicative fitness, whereas when Ne was high (i.e. low genetic drift), the final PVY replicative fitness was high independently of the replicative fitness of the initially inoculated virus. We show that combining a high resistance efficiency (low Wi) and a strong genetic drift (low Ne) is the best solution to increase resistance durability, that is, to avoid virus adaptation on the long term.

Comparative Genomics Screens Identify a Novel Small Secretory Peptide, SlSolP12, which Activates Both Local and Systemic Immune Response in Tomatoes and Exhibits Broad-Spectrum Activity.

Small secreted peptides (SSPs) are essential for defense mechanisms in plant-microbe interactions, acting as danger-associated molecular patterns (DAMPs). Despite the first discovery of SSPs over three decades ago, only a limited number of SSP families, particularly within Solanaceae plants, have been identified due to inefficient approaches. This study employed comparative genomics screens with Solanaceae proteomes (tomato, tobacco, and pepper) to discover a novel SSP family, SolP. Bioinformatics analysis suggests that SolP may serve as an endogenous signal initiating the plant PTI response. Interestingly, SolP family members from tomato, tobacco, and pepper share an identical sequence (VTSNALALVNRFAD), named SlSolP12 (also referred to as NtSolP15 or CaSolP1). Biochemical and phenotypic analyses revealed that synthetic SlSolP12 peptide triggers multiple defense responses: ROS burst, MAPK activation, callose deposition, stomatal closure, and expression of immune defense genes. Furthermore, SlSolP12 enhances systemic resistance against Botrytis cinerea infection in tomato plants and interferes with classical peptides, flg22 and Systemin, which modulate the immune response. Remarkably, SolP12 activates ROS in diverse plant species, such as Arabidopsis thaliana, soybean, and rice, showing a broad spectrum of biological activities. This study provides valuable approaches for identifying endogenous SSPs and highlights SlSolP12 as a novel DAMP that could serve as a useful target for crop protection.

Comparative chloroplast genome analyses of cultivated and wild Capsicum species shed light on evolution and phylogeny.

The chloroplast (cp.) genome, also known as plastome, plays crucial roles in plant survival, adaptation, and evolution. The stable genetic structure of cp. genomes provides an ideal system for investigating species evolution. We sequenced three complete cp. genome sequences of Capsicum species and analyzed them using sequences of various Capsicum species retrieved from the NCBI database. The cp. genome of Capsicum species maintains a well-preserved quadripartite structure consisting of two inverted repeats (IRs) flanked by a large single copy (LSC) region and a small single copy (SSC) region. The sizes of cp. genome sequences ranged from 156,583 bp (C. lycianthoides) to 157,390 bp (C.pubescens). A total of 127-132 unique genes, including 83-87 protein-coding, 36-37 tRNA, and eight rRNA genes, were predicted. Comparison of cp. genomes of 10 Capsicum species revealed high sequence similarity in genome-wide organization and gene arrangements. Fragments of trnT-UGU/trnL-UAA, ccsA, ndhD, rps12, and ycf1 were identified as variable regions, and nucleotide variability of LSC and SSC was higher than that of IR. Phylogenetic speciation analysis showed that the major domesticated C. annuum species were the most extensively divergent species and closely related to C. tovarii and C. frutescens. Analysis of divergent times suggested that a substantial range of speciation events started occurring ~ 25.79 million years ago (Mya). Overall, comparative analysis of cp. genomes of Capsicum species not only offers new insights into their genetic variation and phylogenetic relationships, but also lays a foundation for evolutionary history, genetic diversity, conservation, and biological breeding of Capsicum species.

Fine Mapping and Candidate Gene Analysis of Two Major Quantitative Trait Loci, qFW2.1 and qFW3.1, Controlling Fruit Weight in Pepper (Capsicum annuum).

Fruit weight is an important agronomic trait in pepper production and is closely related to yield. At present, many quantitative trait loci (QTL) related to fruit weight have been found in pepper; however, the genes affecting fruit weight remain unknown. We analyzed the fruit weight-related quantitative traits in an intraspecific Capsicum annuum cross between the cultivated species blocky-type pepper, cv. Qiemen, and the bird pepper accession, "129-1" (Capsicum annuum var. glatriusculum), which was the wild progenitor of C. annuum. Using the QTL-seq combined with the linkage-based QTL mapping approach, QTL detection was performed; and two major effects of QTL related to fruit weight, qFW2.1 and qFW3.1, were identified on chromosomes 2 and 3. The qFW2.1 maximum explained 12.28% of the phenotypic variance observed in two F2 generations, with the maximum LOD value of 11.02, respectively; meanwhile, the qFW3.1 maximum explained 15.50% of the observed phenotypic variance in the two F2 generations, with the maximum LOD value of 11.36, respectively. qFW2.1 was narrowed down to the 1.22 Mb region using homozygous recombinant screening from BC2S2 and BC2S3 populations, while qFW3.1 was narrowed down to the 4.61Mb region. According to the transcriptome results, a total of 47 and 86 differentially expressed genes (DEGs) in the candidate regions of qFW2.1 and qFW3.1 were identified. Further, 19 genes were selected for a qRT-PCR analysis based on sequence difference combined with the gene annotation. Finally, Capana02g002938 and Capana02g003021 are the most likely candidate genes for qFW2.1, and Capana03g000903 may be a candidate gene for qFW3.1. Taken together, our results identified and fine-mapped two major QTL for fruit weight in pepper that will facilitate marker-assistant breeding for the manipulation of yield in pepper.

Developing an Optimized Protocol for Regeneration and Transformation in Pepper.

Capsicum annuum L. is extensively cultivated in subtropical and temperate regions globally, respectively, when grown in a medium with 8 holding significant economic importance. Despite the availability of genome sequences and editing tools, gene editing in peppers is limited by the lack of a stable regeneration and transformation method. This study assessed regeneration and transformation protocols in seven chili pepper varieties, including CM334, Zunla-1, Zhongjiao6 (ZJ6), 0818, 0819, 297, and 348, in order to enhance genetic improvement efforts. Several explants, media compositions, and hormonal combinations were systematically evaluated to optimize the in vitro regeneration process across different chili pepper varieties. The optimal concentrations for shoot formation, shoot elongation, and rooting in regeneration experiments were determined as 5 mg/L of 6-Benzylaminopurine (BAP) with 5 mg/L of silver nitrate (AgNO3), 0.5 mg/L of Gibberellic acid (GA3), and 1 mg/L of Indole-3-butyric acid (IBA), respectively. The highest regeneration rate of 41% was observed from CM334 cotyledon explants. Transformation optimization established 300 mg/L of cefotaxime for bacterial control, with a 72-h co-cultivation period at OD600 = 0.1. This study optimizes the protocols for chili pepper regeneration and transformation, thereby contributing to genetic improvement efforts.

Characterization of GPX Gene Family in Pepper (Capsicum annuum L.) under Abiotic Stress and ABA Treatment.

Plant glutathione peroxidases (GPXs) are important enzymes for removing reactive oxygen species in plant cells and are closely related to the stress resistance of plants. This study identified the GPX gene family members of pepper (Capsicum annuum L.), "CM333", at the whole-genome level to clarify their expression patterns and enzyme activity changes under abiotic stress and ABA treatment. The results showed that eight CaGPX genes were unevenly distributed across four chromosomes and one scaffold of the pepper genome, and their protein sequences had Cys residues typical of the plant GPX domains. The analysis of collinearity, phylogenetic tree, gene structure, and conserved motifs indicated that the CaGPX gene sequence is conserved, structurally similar, and more closely related to the sequence structure of Arabidopsis. Meanwhile, many cis elements involved in stress, hormones, development, and light response were found in the promoter region of the CaGPX gene. In addition, CaGPX1/4 and CaGPX6 were basically expressed in all tissues, and their expression levels were significantly upregulated under abiotic stress and ABA treatment. Subcellular localization showed that CaGPX1 and CaGPX4 are localized in chloroplasts. Additionally, the variations in glutathione peroxidase activity (GSH-Px) mostly agreed with the variations in gene expression. In summary, the CaGPXs gene may play an important role in the development of peppers and their response to abiotic stress and hormones.

Genomic structure and marker-trait association for plant and fruit traits in Capsicum chinense and Capsicum baccatum germplasm.

OBJECTIVES: Capsicum baccatum and C. chinense are domesticated pepper species originating from Latin America recognized for their unique flavor and taste and widely diffused as spicy food for fresh uses or for processing. Owing to their capacity for adaptation to diverse habitats in tropical regions, these species serve as a valuable resource for agronomic traits and tolerance to both biotic and abiotic challenges in breeding projects. This study aims to dissect the genetic diversity of C. baccatum and C. chinense germplasm and to detect candidate genes underlying the variation of plant morphological and fruit size and shape traits. To that goal, SNP data from genotyping by sequencing have been used to investigate the genetic diversity and population structure of 103 accessions belonging to the two species. Further, plants have been assessed with main plant descriptors and fruit imaging analysis and association between markers and traits has been performed. RESULTS: The population structure based on 29,820 SNPs revealed 4 subclusters separating C. chinense and C. baccatum individuals. A deeper analysis within each species highlighted three subpopulations in C. chinense and two in C. baccatum. Phenotypic characterization of 54 traits provided approximately 125 thousand datapoints highlighting main differences between species for flower and fruit traits rather than plant architecture. Marker-traits association, performed with the CMLM model, revealed a total of 6 robust SNPs responsible for change in flower traits and fruit shape. This is the first attempt for mapping morphological traits and fruit features in the two domesticated species, paving the way for further genomic assisted breeding.

Highly efficient CRISPR/Cas9-RNP mediated CaPAD1 editing in protoplasts of three pepper (Capsicum annuum L.) cultivars.

Parthenocarpy, characterized by seedless fruit development without pollination or fertilization, offers the advantage of consistent fruit formation, even under challenging conditions such as high temperatures. It can be induced by regulating auxin homeostasis; PAD1 (PARENTAL ADVICE-1) is an inducer of parthenocarpy in Solanaceae plants. However, precise editing of PAD1 is not well studied in peppers. Here, we report a highly efficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) for CaPAD1 editing in three valuable cultivars of pepper (Capsicum annuum L.): Dempsey, a gene-editable bell pepper; C15, a transformable commercial inbred line; and Younggo 4, a Korean landrace. To achieve the seedless pepper trait under high temperatures caused by unstable climate change, we designed five single guide RNAs (sgRNAs) targeting the CaPAD1 gene. We evaluated the in vitro on-target activity of the RNP complexes in three cultivars. Subsequently, we introduced five CRISPR/Cas9-RNP complexes into protoplasts isolated from three pepper leaves and compared indel frequencies and patterns through targeted deep sequencing analyses. We selected two sgRNAs, sgRNA2 and sgRNA5, which had high in vivo target efficiencies for the CaPAD1 gene across the three cultivars and were validated as potential off-targets in their genomes. These findings are expected to be valuable tools for developing new seedless pepper cultivars through precise molecular breeding of recalcitrant crops in response to climate change.

Efficient In Vitro Regeneration System and Comparative Transcriptome Analysis Offer Insight into the Early Development Characteristics of Explants from Cotyledon with Partial Petiole in Small-Fruited Pepper (Capsicum annuum).

In our research, we utilized six small-fruited pepper germplasms as materials, selected cotyledons with the petiole and hypocotyls as explants, and conducted in vitro regeneration studies. Our outcomes specify that the most suitable explant is cotyledon with the petiole, and the suitable genotype is HNUCA341. The optimal medium for inducing and elongating adventitious buds for this genotype is Murashige and Skoog medium (MS) + 9.12 µM Zeatin (ZT) + 0.57 µM 3-Indoleacetic acid (IAA), with a bud induction rate of 44.4%. The best rooting induction medium is MS + 1.14 µM IAA, with a rooting rate of 86.7%. Research on the addition of exogenous hormones has revealed that the induction speed of buds in small-fruited pepper (HNUCA341) in the combination of ZT and IAA hormones (abbreviated as ZI) is quicker, and the induction effect is better. The histological observations indicate that ZI treatment accelerates the initiation of explant division and differentiation, causing a shorter duration of vascular-bundle tissue production. The plant hormone signaling pathway was significantly enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, including ARR9 (LOC107843874, LOC107843885), ARR4 (LOC107848380, LOC107862455), AHK4 (LOC107870540), AHP1 (LOC107839518), LAX2 (LOC107846008), SAUR36 (LOC107852624), IAA8 (LOC107841020), IAA16 (LOC107839415), PYL4 (LOC107843441), and PYL6 (LOC107871127); these significantly enriched genes may be associated with in vitro regeneration. In addition, the carbon metabolism pathway and plant mitogen-activated protein kinase (MAPK) signaling pathway are also significantly enriched in KEGG. The results of the Gene Ontology (GO) analysis revealed that differentially expressed genes related to carbon metabolism and fixation, photosynthesis and MAPK signaling pathways were upregulated under ZI treatment. It was found that they might be associated with enhanced regeneration in vitro. Furthermore, we also screened out differentially expressed transcription factors, primarily from the MYB, bHLH, AP2/ERF, and NAC families. Overall, our work accumulated important data for the in-depth analysis of the molecular mechanism of in vitro regeneration of pepper, and provides valuable germplasm for establishing an efficient stable pepper genetic-transformation system based on tissue culture.

Dissection of Metabolome and Transcriptome-Insights into Capsaicin and Flavonoid Accumulation in Two Typical Yunnan Xiaomila Fruits.

Pepper is an economically important vegetable worldwide, containing various specialized metabolites crucial for its development and flavor. Capsaicinoids, especially, are genus-specialized metabolites that confer a spicy flavor to Capsicum fruits. In this work, two pepper cultivars, YB (Capsicum frutescens L.) and JC (Capsicum baccatum L.) pepper, showed distinct differences in the accumulation of capsaicin and flavonoid. However, the molecular mechanism underlying them was still unclear. Metabolome analysis showed that the JC pepper induced a more abundant accumulation of metabolites associated with alkaloids, flavonoids, and capsaicinoids in the red ripening stages, leading to a spicier flavor in the JC pepper. Transcriptome analysis confirmed that the increased expression of transcripts associated with phenylpropanoid and flavonoid metabolic pathways occurred in the JC pepper. Integrative analysis of metabolome and transcriptome suggested that four structural genes, 4CL7, 4CL6, CHS, and COMT, were responsible for the higher accumulation of metabolites relevant to capsaicin and flavonoids. Through weighted gene co-expression network analyses, modules related to flavonoid biosynthesis and potential regulators for candidate genes were identified. The promoter analysis of four candidate genes showed they contained several cis-elements that were bonded to MYB, bZIP, and WRKY transcription factors. Further RT-qPCR examination verified three transcription factors, MYB, bZIP53, and WRKY25, that exhibited increased expression in the red ripening stage of the JC pepper compared to YB, which potentially regulated their expression. Altogether, our findings provide comprehensive understanding and valuable information for pepper breeding programs in the future.

Differential responses of bell pepper genotypes to indigenous Pseudomonas putida A32 treatment: implications for drought resilience.

AIMS: This study aimed to evaluate the potential of endophytic plant growth-promoting bacterium (PGPB), Pseudomonas putida A32, to mitigate drought stress in two bell pepper genotypes, Amfora 19 and Amfora 26, and to assess the genotype-specific responses to bacterial treatment. METHODS AND RESULTS: The isolate P. putida A32 was selected for its remarkable beneficial properties, exhibiting 13 out of 14 traits tested. Under drought conditions, Amfora 26 showed increased relative water content and decreased H2O2 and malondialdehyde following bacterial treatment, while Amfora 19 exhibited enhanced growth parameters but responded less to bacterial treatment regarding drought parameters. However, Amfora 19 displayed inherent drought tolerance mechanisms, as indicated by lower stress parameters compared to Amfora 26. CONCLUSIONS: The study emphasizes the importance of genotype-specific responses to PGPB treatment and the mechanisms of drought tolerance in peppers. Pseudomonas putida A32 effectively mitigated drought stress in both genotypes, with differential responses influenced by plant genotype. Our study confirmed our initial hypothesis that Amfora 19, as a genotype tolerant to biotic stress, is also more tolerant to abiotic stress. Understanding these interactions is crucial for the development of customized strategies to improve plant productivity and tolerance to drought.

Estimation of environment stability for fruit yield and capsaicin content by using two models in Capsicum chinense Jacq. (Ghost Pepper) with multi-year evaluation.

Background: Capsicum chinense Jacq. (Ghost Pepper) is well-known for its high pungency and pleasant aroma. The recent years witnessed a significant decline in popularity of this important crop due to the use of inferior planting material and lack of elite lines. To maintain constant performance across a variety of settings, it is crucial to choose stable lines with high yield and capsaicin content, as these are the most promising traits of Ghost Pepper. Method: In this study, 120 high-capsaicin genotypes were subjected to a 3-year (kharif 2017, 2018 and 2019) stability investigation utilizing two well-known stability methods: Eberhart-Russell (ER) and additive main effects and multiple interaction (AMMI). Three replications were used following Randomized Complete Block Design for 11 traits. The experiment soil was sandy loam with pH 4.9. Minimum and maximum temperature of 18.5 °C, 17.5 °C, 17.4 °C and 32.2 °C, 31.3 °C, 32.7 °C and rainfall of 1,781, 2,099, 1,972 mm respectively was recorded for the study period. Result: The genotype-environment linear interaction (G×E Lin.) was highly significant for days to 50% flowering, capsaicin content, fruit length and girth, fruit yield per plant and number of fruits per plant at p < 0.005. G×E interaction for fruit yield and capsaicin content in AMMI-analysis of variance reported 67.07% and 71.51% contribution by IPCA-1 (interactive principal component axis) and 32.76% and 28.49% by IPCA-2, respectively. Eight genotypes were identified to be stable with high yield and capsaicin content. The identified stable lines can be opted for cultivation to reduce the impact of crop failure when grown in different macro-environments. Moreover, the pharmaceutical and spice sectors will also be benefitted from the lines with high capsaicin content. Further research assessing the lines' performance across various regions of India can provide a solid foundation for the crop's evaluation at national level.

Insights into bs5 resistance mechanisms in pepper against Xanthomonas euvesicatoria through transcriptome profiling.

BACKGROUND: Bacterial spot of pepper (BSP), caused by four different Xanthomonas species, primarily X. euvesicatoria (Xe), poses a significant challenge in pepper cultivation. Host resistance is considered the most important approach for BSP control, offering long-term protection and sustainability. While breeding for resistance to BSP for many years focused on dominant R genes, introgression of recessive resistance has been a more recent focus of breeding programs. The molecular interactions underlying recessive resistance remain poorly understood. RESULTS: In this study, transcriptomic analyses were performed to elucidate defense responses triggered by Xe race P6 infection by two distinct pepper lines: the Xe-resistant line ECW50R containing bs5, a recessive resistance gene that confers resistance to all pepper Xe races, and the Xe-susceptible line ECW. The results revealed a total of 3357 upregulated and 4091 downregulated genes at 0, 1, 2, and 4 days post-inoculation (dpi), with the highest number of differentially expressed genes (DEGs) observed at 2 dpi. Pathway analysis highlighted DEGs in key pathways such as plant-pathogen interaction, MAPK signaling pathway, plant hormone signal transduction, and photosynthesis - antenna proteins, along with cysteine and methionine metabolism. Notably, upregulation of genes associated with PAMP-Triggered Immunity (PTI) was observed, including components like FLS2, Ca-dependent pathways, Rboh, and reactive oxygen species (ROS) generation. In support of these results, infiltration of ECW50R leaves with bacterial suspension of Xe led to observable hydrogen peroxide accumulation without a rapid increase in electrolyte leakage, suggestive of the absence of Effector-Triggered Immunity (ETI). Furthermore, the study confirmed that bs5 does not disrupt the effector delivery system, as evidenced by incompatible interactions between avirulence genes and their corresponding dominant resistant genes in the bs5 background. CONCLUSION: Overall, these findings provide insights into the molecular mechanisms underlying bs5-mediated resistance in pepper against Xe and suggest a robust defense mechanism in ECW50R, primarily mediated through PTI. Given that bs5 provides early strong response for resistance, combining this resistance with other dominant resistance genes will enhance the durability of resistance to BSP.

CaAOS as a hub gene based on physiological and transcriptomic analyses of cold-resistant and cold-sensitive pepper cultivars.

The yield and quality of pepper are considerably influenced by the cold conditions. Herein, we performed morphological, physiological and transcriptomic analyses by using two pepper seedlings, '2379' (cold-resistant) and '2380' (cold-sensitive). Briefly, 60 samples from each cultivar were analyzed at four distinct time points (0, 6, 24 and 48 h) at 5 °C in darkness. The physiological indices and activities of enzymes exhibited marked differences between the two cultivars. Transcriptomic analysis indicated that, compared to the control group, 11,415 DEGs were identified in '2379' and '2380' at 24 h. In the early stage, the number of DEGs in '2379' was 5.68 times higher than that in '2380', potentially explaining the observed differences in tolerance to colds. Processes such as protein targeting to membranes, jasmonic acid (JA)-mediated signalling, cold response and abscisic acid-activated signalling were involved. Subsequently, we identified a hub gene, CaAOS, that is involved in JA biosynthesis, positively influences cold tolerance and is a target of CaMYC2. Variations in the GC-motif of the CaAOS's promoter may influence the expression levels of CaAOS under cold treatment. The result of this study may lead to the development of more effective strategies for enhancing cold tolerance, potentially benefitting pepper breeding in cold regions.

Functional analysis of the CaPIPLC5 gene in the regulation of the fertility restoration in pepper.

Cytoplasmic male sterility (CMS) is a very important factor to produce hybrid seeds, and the restoration of fertility involves the expression of many fertility-related genes. Our previous study showed that the expression of CaPIPLC5 was significantly up-regulated in pepper restorer accessions and minimally expressed in sterile accessions, speculating that CaPIPLC5 is related to the restoration of fertility. In this study, we further validated the function of CaPIPLC5 in the restoration of fertility. The results showed that CaPIPLC5 was specifically expressed in the anthers of the restorer accessions with the subcellular localization in the cytoplasm. Furthermore, the expression of CaPIPLC5 was significantly higher in restorer lines and restorer combinations than that in CMS lines and their maintainer lines. Silencing CaPIPLC5 led to the number of pollen decreased, pollen grains wrinkled, and the ratio of pollen germination reduced. In addition, the joint analysis of Yeast One-Hybrid (Y1H) and Dual-Luciferase (dual-LUC) assays suggested that transcription factors such as CaARF5, CabZIP24 and CaMYB-like1, interacted with the promoter regions of CaPIPLC5, which regulated the expression of CaPIPLC5. The present results provide new insights into the study of CaPIPLC5 involved in the restoration of fertility in pepper.

Silicon improves the drought tolerance in pepper plants through the induction of secondary metabolites, GA biosynthesis pathway, and suppression of chlorophyll degradation.

Drought stress caused by the global climate considerably disturbs plant yield and growth. Here, we explored the putative roles of silicon in repressing drought mechanisms in pepper and the prominent involvement of secondary metabolites, GA pathway, and photosystem II. Our research revealed that the transcript level of the flavonoid biosynthesis-associated genes, including the PAL, 4-CL, CHS, FLS-1, F3H and DFR, progressively induced in the pepper leaves treated with silicon during the drought stress duration. Moreover, the phenolic and flavonoid compounds extensively induced in the pepper plants. Furthermore, the pepper plants markedly inhibited chlorophyll catabolic-allied genes, senescence-related marker gene, and the Rbohs gene. Silicon application also sustained the membrane stability, supported via fewer electrolyte leakage processes and minor, O2- H2O2 and MDA levels during drought. Apart from this, the pepper plants significantly induced the expression level of the photosystem II-related genes, osmoprotectants pathway-associated genes, and antioxidant defense genes. Moreover, the GA biosynthesis genes were prompted, while the ABA signaling and biosynthesis genes were suppressed in the silicon-supplemented plants. These consequences infer that the role of Si supplementation on enhancing drought tolerance could be elucidated through the activation of secondary metabolites, flavonoid biosynthesis, osmoprotectants, GA pathway, the efficiency of PSII, and the suppression of chlorophyll degradation. Our research outcomes unveil new and remarkable characteristics of silicon supplementation and offer a series of candidate targets for improving the tolerance of pepper plants to drought stress.

Characterization of the GGP gene family in potato (Solanum tuberosum L.) and Pepper (Capsicum annuum L.) and its expression analysis under hormonal and abiotic stresses.

GDP-L-galactose phosphorylase (GGP) is a key rate-limiting enzyme in plant ascorbic acid synthesis, which plays an important role in plant growth and development as well as stress response. However, the presence of GGP and its function in potato and pepper are not known. In this study, we first identified two GGP genes in each potato and pepper genomes using a genome-wide search approach. We then analyzed their physicochemical properties, conserved domains, protein structures and phylogenetic relationships. Phylogenetic tree analysis revealed that members of the potato and pepper GGP gene families are related to eggplant (Solanum melongena L.), Arabidopsis (Arabidopsis thaliana L.), tobacco (Nicotiana tabacum L.) and tomato (Solanum lycopersicum L.), with tomato being the most closely related. The promoter sequences mainly contain homeopathic elements such as light-responsive, hormone-responsive and stress-responsive, with light-responsive elements being the most abundant. By analyzing the structure of the genes, it was found that there is no transmembrane structure or signal peptide in the GGP gene family of potatoes and peppers, and that all of its members are hydrophilic proteins. The expression profiles of different tissues show that StGGP1 has the highest expression levels in leaves, StGGP2 has the highest expression levels in stamens, and CaGGPs have the highest expression levels in the early stages of fruit development (Dev1). It was found that StGGPs and CaGGPs genes showed different response to phytohormones and abiotic stresses. Abscisic acid (ABA) treatment induced the most significant change in the expression of StGGPs, while the expression of CaGGPs showed the most pronounced change under methyl jasmonate (MeJA) treatment. StGGPs responded mainly to dark treatment, whereas CaGGPs responded mainly to NaCl stress. These results provide an important basis for a detailed study about the functions of GGP homologous genes in potato and pepper in response to abiotic stresses.

A comprehensive physiological and -Omic analysis of trypsin-mediated protection of green pepper fruits from chilling injury.

Chilling injury (CI) in green pepper fruits during low-temperature storage causes a significant decline in quality. The present study utilized physiological, transcriptomic, and metabolomic analyses to idneitfy the mechanisms by which trypsin mitigates CI in green peppers stored at 4 °C for 8 days, followed by 3 days of shelf life. Results indicated that the trypsin treatment significantly reduced electrolyte leakage and the CI index in peppers, effectively extending their shelf life and preserving postharvest quality. After 4 days of storage, comparative -omic analyses identified 2514 differentially expressed genes (DEGs) and 397 differentially abundant metabolites (DAMs) between trypsin-treated and control peppers. The trypsin treatment induced changes in sugar metabolism, modulating the expression of HK, SUS, INV, and GLGC, which affected the abundance of metabolites such as CDP-glucose and α-D-p-glucose. Trypsin also enhanced carotenoid metabolism, altering the abundance of rhodopinal glucoside, 1'-hydroxyl-γ-carotene glucoside, and farnesyl 1-PP, and influencing the expression of PDS, CRTH, CRTB, and LUT5. Notably, the trypsin treatment activated the mitogen-activated protein kinase (MAPK) pathway that plays an integral role in the signal transduction of abiotic stress. Differential expression of FLS2, ELF18, PTO, PR1, PTI5, WPKY, MEKK1, and MPK6 genes in the MAPK pathway was observed, which was correlated with CI mitigation in green peppers during cold storage. In conclusion, trypsin is an effective treatment for reducing CI in green peppers during cold storage. The present study provides valuable insights into its physiological and molecular impact on green pepper fruit.